The present application relates to a system and methods for quantifying differences in measured values within histograms, and more specifically, for automation of calibration of a flow cytometer (or other fluorescence-based instrument) based on construction and use of a distance function having a bin-to-bin dissimilarity matrix, to determine a statistical significance of distance between histograms, and reproducibility of measurements performed on flow cytometry systems.
Flow cytometry is a general technique for counting, examining, and sorting large numbers of microscopic particles suspended in a stream of fluid. It allows simultaneous multiparametric analysis of the physical and/or chemical characteristics of single particles flowing through an optical and electronic detection apparatus using light scattering, fluorescence, and absorbance measurements.
Modern flow cytometers are able to analyze several thousand particles every second, in “real time,” and can actively separate and isolate particles having specified properties. A flow cytometer is similar to a microscope, except that instead of producing an image of the cell, flow cytometry offers “high-throughput” (for a large number of cells) automated quantification of singular values of light scattering, fluorescence or absorbance (integrated over particle volume).
FIG. 1 is a flow diagram of a simplified, conventional flow cytometer 10. The flow cytometer 10 includes a laser 14, a flow cell 16, an optical system including a collection lens 20, a beam splitter 24, a dichroic mirror 28, and a number of optical filters 32. A dichroic mirror is used to reflect light selectively according to a specific wavelength. Accordingly, multiple dichroic mirrors 28 may be used to attempt to direct light of a specific wavelength. The flow cytometer 10 further includes detectors, including a forward-scatter detector 36, a side-scatter detector 40, one or more fluorescence detectors 44, and an absorbance detector (not shown). The absorbance detector, if included, would be aligned in line with the laser beam and would detect loss in axial light, indicating an absorbance thereof. An amplification system (not shown) may also be employed, wherein amplifiers are placed after the detectors to strengthen signals of detected scattered light or fluorescence.
The laser 14 emits excitation light, which is directed onto the flow cell 16, which includes a hydro-dynamically focused stream of fluid having the sample of interest. More specifically, the flow cell is a glass, quartz or a plastic piece of fluidic equipment enclosing a stream of sheath fluid, which carries particles. The point of focus of the laser beam within the flow cell 16 is referred to as the interrogation point. The excitation light can come from another source besides a laser. The light scattering and/or fluorescence emission occurs upon illumination (irradiation) of the excitation light, which then passes through the optical system components listed above, depending on the wavelength corresponding to the individual photons that have been excited and their direction of travel.
The detectors listed above are aimed at the point where the fluid stream passes through the light beam; one in line with the light beam (Forward Scatter or FSC) 36, several perpendicular to it (Side Scatter (SSC) 40, and one or more fluorescence detectors 44). Each suspended particle (from 0.2 to 150 micrometers in diameter) passing through the beam scatters the light in some way, and fluorescent chemicals found in the particle or attached to the particle may be excited into emitting luminescence (fluorescence, phosphorescence) or Raman signal. This combination of scattered (or transmitted) and fluorescent light is picked up by the detectors, wherein the scattered light is detected by the forward and side scatter detectors 36, 40 and the emitted light is detected by the fluorescence detectors 44.
The detectors are connected to a computer (FIG. 2), which analyzes the intensity of the light incident at each detector. By analyzing intensities of collected signals at each detector, it is then possible to derive various types of information about the physical and chemical structure of each individual particle of the fluid sample. For instance, FSC correlates with the cell volume and SSC depends on the inner complexity of the particle (i.e. shape of the nucleus, the amount and type of cytoplasmic granules or the membrane roughness).
In summary, and further to different embodiments, a flow cytometer has five main components: (1) the flow cell: includes liquid stream (sheath fluid) to carry and align the particles so that they pass single file through the light beam for sensing; (2) the optical system having illumination sources: lamps (mercury, xenon); high power water-cooled lasers (argon, krypton, dye laser); low power air-cooled lasers (argon (488 nm), red-HeNe (633 nm), green-HeNe, HeCd (UV)); diode lasers (blue, green, red, violet) resulting in light signals; (3) the detectors (typically photomultipliers or avalanche photodiodes) and an Analog-to-Digital Conversion (ADC) system: converting FSC and SSC as well as fluorescence signals from light into electrical signals that can be processed by a computer; (4) the amplification system: linear or logarithmic; and (5) the computer for analysis of the signals. Early flow cytometers were generally experimental devices, but recent technological advances have created a considerable market for the instrumentation, as well as the reagents used in analysis, such as fluorescently-labeled antibodies, calibration beads, and analysis software.
Flow cytometric assays have been developed to determine both cellular characteristics such as size, membrane potential, and intracellular pH, and the levels of cellular components such as DNA, protein, and surface receptors. Measurements in flow cytometry are presented for further interpretation and analysis as distributions of parameters measured in population of cells.
Flow cytometers must be calibrated prior to quantitative fluorescence intensity measurements because variability and drift in detectors, as well as variability of the environment (e.g., changes in ambient temperature). To account for this possible variability, a well-known and well-characterized set of bioparticles is usually analyzed using the cytometry instrument prior to actual experiments. Well-defined characteristics include size, and thus light scatter properties, as well as fluorescence properties. One example of bioparticles that could be used for calibration includes fixed chicken red blood cells. Subsequently, the photomultiplier tube (PMT) voltages of the cytometer are adjusted accordingly to place the peak fluorescence distribution representing the standard into predetermined bins of cytometry histograms. Cells can be substituted by standardized calibration beads, which carry fluorescent labels bound on the surface (e.g., fluorescein, PE, etc). In either case, all the data collected after this calibration process are reported as values relative to the intensity of the standard sample.
The calibration procedure can be further enhanced by use of molecules of equivalent soluble fluorophore (MESF)-calibrated beads. The MESF value of a bead corresponds to the number of fluorescent molecules in solution which give equal fluorescence to that of the bead, to which the same molecules are bound. MESF calibration can be used in flow cytometry as well as in other fluorescence-based methods, such as micro-array readers. The assignments of MESF values to a set of labeled beads with different fluorophore densities allow for construction of a calibration curve for an instrument. The procedure allows one to obtain MESF values of biological cells stained with the same fluorescent tag (e.g., fluorophore-labeled monoclonal antibodies). However, this calibration does not take under consideration the fact that with lower amounts of fluorescent labels of surface of beads (or cells), uncertainty of measurement increases: the corresponding histogram peak becomes wider. Therefore, despite calibration, a comparison of samples representing low and high fluorescence intensities cannot be performed in a statistically relevant manner. In other words, statistical significance cannot be assigned.
Flow-cytometry calibration beads are manufactured by a dozen different manufacturers. Calibration of a cytometer has to be performed daily while in use to ensure proper results of live samples. In some clinical systems, this calibration is required to be performed more than once per day. This is not only a question of good practices, but an actual requirement.
The whole calibration process is performed manually, such as described in recent literature. See, e.g., Robert A. Hoffman and James C. S. Wood, Characterization of Flow Cytometer Instrument Sensitivity, Current Protocols in Cytometry, 1.20.1-1.20.18 (2007) (describing linear regression calculations to be made with the aid of a spreadsheet); Robert A. Hoffman, Standardization, Calibration, and Control in Flow Cytometry, Current Protocols in Cytometry, 1.3.1-1.3.21 (2005). Dr. Hoffman in the second reference listed above quotes from the Clinical Laboratory Standards Institute, an international clinical laboratory standards-setting organization, which stated that “[t]here are no standards which can be used to check the accuracy of flow cytometric test results. Hence, verifying reproducibility of instrument performance is an essential element of daily quality assurance for the flow cytometry laboratory. Instrument performance must be monitored under the same conditions as are used to run test samples.” Id. The present disclosure is aimed at ameliorating this lack of standards in checking the accuracy and reproducibility of flow cytometric results.
There exist about 10 thousand clinical analyzers, and about 20-30 thousand research machines in the field for testing. Additionally, there are at least five thousand flow cytometry cell sorters that are calibrated also with the manual, bead method. Accordingly, there is a tremendous need to provide a system and methods to, in an automated fashion, validate measured values of a cytometer, or other fluorescence-based measuring instrument, wherein a standard is provided, outside of which variation in measured values render test results unreliable. This would provide a quality control system that would save a significant amount of time in guess work involved when a cytometer operator is faced with larger-than-normal variations in measured values.